<p>This famous quote from Richard Feynman inspired our project. The initial idea of one physicist in our group was: <h3>Let's build a cell!</h3></p>

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<p>So, of course, the biologists of our group tried to explain him, that a cell is far too complex and this is a crazy idea. If a biologist thinks of a cell, he sees an extremely complex structure, but the physicist just sees a simple black circle. So what is a cell, if you break it down to a simpler level?</p>

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<p>A compartment separating a reactive center from the outside, combined with controlled means of transport in and out and some modifications on the outside. An intelligent, microscopically small reactor. And our idea of a </h3>Smart Nanoreactor</h3> was born.</p>

<p> First of all, we needed a compartment. In cells, the compartment is made out of a phospholipid bilayer membrane that forms the plasma membrane. But this being already nature's strategy, we wanted to try a different thing and decided to use block co-polymers to build the nanoreactor wall. </p>

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<!-- <p> The main goal of experiment was to synthesize stable and uniformly distributed polymersomes as vesicle for carrying the collagenase enzyme and also incorporating DNA origami channels for tunable delivery of cargo with respect to its size.</p> -->

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<!-- PMOXA<sub>17</sub>-PDMS<sub>65</sub>-PMOXA<sub>17</sub> was choosen as our ABA tri-block polymer know for its high biocompatiblity and PEG<sub>45</sub>-b-P(DEAEMA<sub>10</sub>-stat-BMA<sub>65</sub>) as our AB di-block polymer which has an additional functional of being pH responsive and also property to encapsulate both hydrophilic and hydrophobic biological entities.</p> -->

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<h3> What are polymers? </h3>

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<p>Polymers consist of structural or repeating units of low molecular weight covalently connected to each other to give high molecular weight compounds. A polymer that consists of two different repeating units is referred to as a co-polymer (Odian, 1981). The covalent linkage of two intrinsically different homo polymers leads to the formation of block–copolymers. Block-copolymers consist of at least two, covalently bound, segments or blocks of different Homo polymers which lead to the formation of di-block or tri-block copolymers. For instance, a tri-block copolymer can have a general form A<sub>n</sub>-B<sub>m</sub>-C<sub>p</sub>, with A, B, C, being different monomer types constituting the different blocks. The subscripts n, m, and p stand for the degree of polymerization, i.e. the average number of each monomer present in each respective block (McGrath, 1981).</p>

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<h3>Why using polymers for our Nanoreactor?</h3>

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<p>There is growing interest in polymersomes due to their potential application in biotechnology and pharmacology (Blanazs et al., 2009) Currently, a great potential lies in their ability to mimic biological membranes by establishing aqueous three dimensional functional cavities. The stability of these membranes is higher than those of biological lipid-based vesicles. Thus, polymersomes consisting of amphiphilic block copolymers hold promise as alternative vesicles in biotechnological and biomedical applications (van Dongen et al., 2011).</p>

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<p> Two important parameters of the membranes are their permeability and their stability. The advantage of synthetic block-copolymers as the building blocks of these nanoreactors is their higher stability over lipids due to the increased length, conformational freedom, and slower dynamics of the underlying polymers.</p>

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<p>Other desired characteristics for the Nanoreactor the polymers can achieve are:

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<ul>

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<li> The shell has a hydrophilic, biocompatible low-protein-binding surface.</li>

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<li> The shell can act as a bio membrane for protein reconstitution.</li>

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<li> The nanovesicle can encapsulate hydrophilic substances.</li>

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<li> The shell material has enormous possibilities for molecular functionalization.</li>

<p>This ABA tri-block copolymer consists of a middle block of PDMS (poly (dimethyl siloxane)) and two PMOXA (poly (2-methyloxazoline)) side chains. These copolymers self-assemble in aqueous solution to form highly stable, closed vesicles with a controlled diameter of typically less than 200 nm.</p>

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</figure>

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<p>Tri-block polymersomes were prepared by film rehydration using PBS buffer (pH 7.4) as rehydrating solvent. The polymersomes were observed using cryo-SEM and cryo-TEM, and the particle size was characterized by DLS (Dynamic light scattering).</p>

<p>Cryo-TEM images clearly depict lamellar and large vesicles formation, which is further confirmed by DLS showing an average diameter of ~950nm and a broad particle size distribution. These are not as desired, so these polymersomes were further subjected to an extrusion process using Ultra-filtration equipment.</p>

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<p> we, the <em>Dresden Nanormous</em>, use polymers to produce hollow nanoreactors or <em>polymersomes</em>, whose walls contain channels for controlled transport. These channels are built by the <em>DNA-origami</em> technique that way we produce a highly flexible system. By encapsulation of an enyzme and the addition of antibodies we create a <em>smart nanoreactor</em> which could be used to <em>fight cancer</em>!

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</div>

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<p> After extrusion, the samples were observed under cryo-TEM again and their size distribution was measured by DLS. Ultra-filtration process extruded larger vesicles and lamellar structure which is well seen in TEM images. In the DLS measurement, the peak of diameter ranging from 226 to 238 nm is better defined, and corresponds to 50% of the total polymersomes.</p>

<a class="fb" rel="polymer" href="http://openwetware.org/images/8/8d/DLS_final.png" title="DLS measurement for tri-block polymersomes. After filtration, the DLS measurement shows a more defined peak for the polymersomes of 200 nm diameter, which correspond to 50 % of the total polymersomes.">

<p>We were also interested in thermo-responsive block copolymer, poly(2-cinnamoylethyl methacrylate)-poly(N-isopropylacryl amide), pH-sensitive and photo-cross-linkable block copolymer. They can function as on/off switches depending on the external pH stimulus due to the pH sensitivity of their cross-linked membrane.</p>

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<p>The block copolymers undergo spontaneous self-assembly into the desired polymersome structures by raising the pH of the aqueous solution from 3 to 10. To preserve the vesicular shape of the polymersomes under varying conditions, cross-linking of their membranes is highly desired.</p>

<p>These di-block polymers self assemble into polymersome of uniform size of 100-120nm and later expand in size till 140 nm as the pH is lowered from alkaline condition to acidic state. Thus, they act as pH responsive polymersomes which can tune its release of biological entities based on environmental pH condition. Such property makes them demandable in field of drug therapy specifically in treating cancer.</p>

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<figcaption><p>Internal reaction</p></figcaption>

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</figure>

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<p>The formation of the polymersomes was analysed using cryo-TEM and the particle size distribution was measured with DLS. In this case, the distribution is very uniform with a peak at 140 nm.</p>

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<p>The pH responsive nature of di-block polymer was investigated by the release profile of Doxorubicin, an anti-cancerogenic agent which showed slow release at pH 7.4 and faster release at pH 5. This is a proof that the polymersomes were effectively cross-linked by UV.</p>

To allow transport inside and outside the smart nanoreactor, a channel is needed. Instead of using a protein channel, we decided to use a DNA origami channel, because it is more robust, and we can easily design it according to the characteristics of the polymer wall.</p>

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<p>

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<h3>What is DNA Origami?</h3>

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</p>

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<p>In DNA origami a circular ssDNA molecule of several thousand base pairs, called scaffold, is folded with the help of short ssDNA strands, the staple strands. The staple strands bring together two different zones of the scaffold, and by varying their sequences different 2D or 3D shapes can be achieved.</p>

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<p>DNA origami has unique properties, such as an addressable surface, which enables selective functionalization with biomolecules and nanomaterials. The origami can also be combined with top-down nanotechnology, such as placement on a fabricated substrate. The technology can also be used in single-molecule imaging, where FRET pairs or fluorophores can be constructed on designed DNA origami structures. Furthermore, DNA origami can be easily combined with other DNA nanodevices such as DNAzymes, DNA beacons or DNA walkers acting on DNA origami. Hence, DNA origami technology has practical potential in various research fields.</p>

<p> The origami channel was designed to be properly integrated into the polymersome wall. For this, the dimensions were restricted to the thickness of the wall, as well as to the length of the scaffold. Furthermore, the dimensions of the porus are determined by the dimensions of the substrate and the enzyme: it has to be big enough so the substrate and products of the reaction can easily go through, but small enough so the enzyme stays encapsulated inside the polymersome. The final dimensions are the following:

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<ul>

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<li>Length: 34 nm</li>

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<li>Diameter: 25 nm</li>

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<li>Pore diameter: 6 nm</li>

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</ul> </p>

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<h3>Scaffold and staple types</h3>

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<p>For the design, the caDNAno program was used (see http://cadnano.org/), and the stability of the structure was tested with CanDo (see http://cando-dna-origami.org/).</p>

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<p>The scaffold used in this design is 7560 bp long, derived from the M13p18 Escherichia coli virus. Its sequence was taken from the cadnano design program. Bending it into a structure with a 78 helix bundles, the desired length is achieved. The channel contains four types of staples:</p>

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<p><ul>

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<li>core: 118 staples give stability to the whole structure.</li>

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<li>edge: 39 staples give stability to the edges.</li>

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<li>helpers: 12 staples have a 5' protruding end with 5 adenines, which should help the anchors point to the right directions.</li>

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<li>anchors: 12 staples have a 3' protruding end of 21 nucleotides (7 nm) which will allow the binding of the oligonucleotides that carry the hydrophobic modifications. Between the corresponding complementary sequence to the scaffold and the protrusion, 5 thymine nucleotides serve as spacer and hybridize with the 5 adenines present in the helpers. </li>

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<li>no_hang: can be used instead of the anchors to generate a channel without any anchors sticking out of the channel walls. They can serve as a negative control when introducing the hydrophobically modified oligonucleotides.</li>

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</ul></p>

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<p>Their sequences and also the map of their distribution in the scaffold can be donwnloaded from the lab book.</p>

<p> The mixture of scaffold and different staples was subjected to a thermal annealing ramp, that allowed the folding. In an initial experiment, different concentrations of Magnesium chloride in the folding buffer were tested, and the resulting structures were analized by atomic force microscopy and electrophoresis to determine the best concentration. The range 10 to 14 mM MgCl<sub>2</sub> were the best, so we chose the 12 mM concentration for further experiments.</p>

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<p>In the AFM images the height and width were measured, obtaining the approximate values of 7 and 60 nm respectively. The height appears to be much lower than the expected, due to the high absorption of the channel to the substrate used for imaging, mica. Mica is negatively charged, and so the Magnesium chloride in the samples interacts with the surface creating a positively charged substrate. Thus, the DNA origami channel can interact tightly with the surface, modifying its structure. The width appears much bigger, which can be due to the tip-sample convolution.</p>

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</div>

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<div class="figure_box">

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<figure class="box " >

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<a class="fb" rel="fold" href="http://openwetware.org/images/4/42/Electro_magchl.png" title="Electrophoresis: Electrophoresis of the channels after folding and filtration using the stated MgCl<sub>2</sub> concentrations in the folding buffer.">

<a class="fb" rel="fold" href="http://openwetware.org/images/3/3f/AFM_magchl.png" title="AFM folding analysis: Corroboration by AFM imaging of the folded or unfolded states of the DNA origami channels after filtration, using folding buffers containing different concentration of MgCl<sub>2</sub>">

<a class="fb" rel="fold" href="http://openwetware.org/images/9/99/AFM_quantification.png" title="AFM measurements: AFM imaging of the folded DNA origami channels after filtration. The graph is the superposition of the height profiles from 20 different structures. From these, the height and width, shown in the table, were calculated. </figcaption>

<p>For the channel to be incorporated inside the polymersome membrane spontaneously, a hydrophobic modification was introduced to a short oligonucleotide that hybridizes with the anchors. Two different types of hydrophobic modifications were performed.</p>

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<h3>5' Palmitate and arachidic acid modification</h3>

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<p>An amino group was introduced to the 5' end of the oligonucleotides, that afterwards reacts with the carboxylic group of the molecule containing the hydrophobic fatty acids palmitate and arachidate. The reaction used is an N-acylation, which requires some activations. More details of the reaction and reagents used can be found in the Lab book.</p>

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<p>The modified oligos were purified with HPLC and the purity of the product was analized by mass spectrometry.</p>

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<h3>3' Cholesterol modification</h3>

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<p>Oligonucleotides for 3' cholesterol modification were synthesized on a solid support already carrying the cholesterol molecules. In this case the sequence was shorter for the modification to be slightly separated from the walls of the channel. The modified oligos were purified with HPLC and the purity of the product was analized with mass spectrometry.</p>

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<h3>Incorporation of the hydrophobically-modified oligonucletides into the origami channel</h3>

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<p> The success of the incorporation of the hydrophobically modified oligos into the DNA origami channel was analyzed by electrophoresis.</p>

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<p/> The DNA origami channel was folded with the presence of no_hang staples, so a structure with no anchors was produced. This channel without anchors was put into contact with the oligonucleotide complementary to the anchors without modification, and also with the oligonucleotide with the two different types of hydrophobic modifications. This served as a negative control as no difference in the electrophoretic mobility should be observed.</p>

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<p> In parallel, the DNA origami channel with the anchor staples was produced in the present of the oligonucleotide complementary to them without the hydrophobic modifications, and the same oligonucleotide with the two different types of modifications</p>

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<p> In the electrophoresis gel a shift can be observed between the electrophoresis mobility of the channels that don't ocntain the anchors and the ones that contain them. This is because the shape of the structure is affected by these sticking out DNA strands in a way that they cannot run as fast in the gel. Moreover, when introducing the oligonucleotides that hybridize with the anchors 5' modified with palmitic and arachidic acid, a further shift is observed, indicating that this modification is correctly incorporated into the channel. As for the 3' cholesterol modification, no shift is observed, but this can be due to the small volume of the cholesterol molecules in respect to the channel and their situation closer to the channel walls. In both modifications, though, a remaining of the sample inside the wells can indicate a certain level of formation of big complexes not able to enter the gel due to aggregation of the channel through their hydrophobic regions, specially in the 5' palmitic and arachidic acid modified channel. This is a good inticative that the hydrophobic modifications were properly incorporated onto the DNA origami channel. </p>

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</div>

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<div class="figure_box">

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<figure class="box " >

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<a class="fb" rel="mod" href="http://openwetware.org/images/0/0c/Purif_palm_arach.png" title="HPLC purification and mass spectrometry analysis of the oligonucleotides modified with palmitate and arachidic acid. A. HPLC chromatogram of the first HPLC purification round. The peak at almost 26 minutes corresponds with the expected elution time of the product, but being so low an elution with methanol was required. B. HPLC chromatogram of the second HPLC purification round, where the product was eluted with methanol. The fraction number 21 was used for the further experiments. C. Mass spectrometry chromatogram of the fraction number 21, the peak corresponds to the product. D. Mass spectrometry spectrum of the amplified peak, the molecular weight of the oligonucleotide with the correct hydrophobic modification is 7270 Da. The highest peak with an m/z of 1815.79 on the right corresponds with the species with 4 positive charges. Other peaks correspond to different number of charges.">

<a class="fb" rel="mod" href="http://openwetware.org/images/a/a3/Purif_chol.png" title="HPLC purification and Mass spectrometry analysis of the oligonucleotides modified with cholesterol. A. HPLC chromatogram. The peak eluting at 10,5 minutes corresponds to the cholesterol-modified oligo. The fraction nr. 36 was used for further experiments. B. Mass spectrometry chromatogram of the fraction number 36, the peak corresponds to the product. D. Mass spectrometry spectrum of the amplified peak, the molecular weight of the oligonucleotide with the cholesterol modification is 5387 Da. The highest peak wit m/z of 1793.86 on the right corresponds with the species with 3 positive charges. Other peaks correspond to different number of charges.">

<a class="fb" rel="mod" href="http://openwetware.org/images/8/88/Electro_hydrophobic_final.png" title="Electrophoresis to analyze if the hydrophobically modified oligonucleotides bind correctly to the anchors in the origami channel.">

<p>The aim of our project is to create a nanoreactor. Inspired by how cell metabolism is organized in specific cellular compartments, we decided to design a system which is capable of hosting enzymatic reactions. Inner compartmentalization allows fragile processes to remain protected against undesired influences, such as proteolytic or microbial degradation and also one could expect enhanced reaction probabilities and efficiencies due to spatial confinement or immobilization of enzymes.</p>

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</div>

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<div class="clear"></div>

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<h2>Choice of reaction</h2>

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<div class="project_box" >

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<p>There are several requirements for the enzyme which can be entrapped. The enzymes should be large enough so that they are not able to escape, especially if there are channels or pores formed in the system. Another requirement is that the reaction is easy to detect. Examples include enzymes which are able to produce fluorescent or chromogenic products, such as myoglobin, Candida Antarctica lipase B, glucose oxides, horse radish peroxidase, urease and α-chymotrypsin. </p>

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<p>The protein of our choice was collagenase. Collagenases are endopeptidases that degrade the helical regions in native collagen preferentially at the Y-Gly bond in the sequence Pro-Y-Gly-Pro, where Y is most frequently a neutral amino acid. This protein is very abundant in many species and it is easy to obtain from the bacteria Clostridium hystoliticum. The isolated enzyme is composed of 7 different proteases ranging in molecular weight from 68-130 kDa, thus the protein is big enough and there is no possibility to pass through the channel. Furthermore, the reaction can be detected easily with spectrophotometric assay.</p>

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</div>

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<div class="clear"></div>

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<h2>Fluorescent labeling</h2>

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<div class="project_box" >

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<p>The protein was conjugated to amine-reactive fluorescent dye in order to prove its entrapment inside the polymersomes. TAMRA NHS was used to perform the fluorescent labeling. TAMRA (Tetramethylrhodamine) belongs to the group of the long-wavelength rhodamines with excitation maximum at 555nm and emission maximum at 580 nm.</p>

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<p>The degree of labelling with TAMRA dye was estimated to be 18 dye molecules per enzyme.</p>

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</div>

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<div class="clear"></div>

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<h2>Enzyme reaction assay</h2>

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<div class="project_box" >

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<p>The enzymatic assay for determining the rate of the reaction was carried out using the peptide FALGPA, standing for N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala. There is sufficient structural similarity between the 2-furanacryoyl group and Pro for FALGPA to be an excellent substrate for collagenase.</p>

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<p>The molecular weight of the substrate is 476 Da. The maximum molecular weight of the molecules that can go through the polymer walls being 400 Da, the substrate will not be able to penetrate the polymer walls, but only to enter the nanoreactor through the channel. Furthermore, the substrate being uncharged, the negative charge of the DNA won't interfere with its transport.</p>

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<p>This peptide has absorbance at 345 nm and the reaction is monitored through continuous spectrophotometric measurement at this wavelength. The assay is made possible by the blue shift in the near-ultraviolet absorption band of the furanacryloyl peptide when the peptide bond between the first and second residues is hydrolyzed, which means that after cleavage of the peptide, the absorbance at 345nm begins to decrease. The enzyme activity before and after conjugation to the dye is characterized through this assay. </p>

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<p>The conjugation to the fluorescent dye leads to a slight decrease in the activity of the enzyme. The FALGPA units are calculated, taking into account the slope of the linear fit. The unmodified enzyme is estimated to have 3 FALGPA units/ml and the conjugated one – 2.6 FALGPA units/ml.</p>

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<p>Since the polymersomes can be source of light scattering events, UPLC measurement of the reaction products was applied for better accuracy of our experimental results.</p>

<a class="fb" rel="rct" href="http://openwetware.org/images/a/a6/Nm_figure2.png" title="Reaction assay with unmodified enzyme. Continuous sprectrophotometric measurement at a wavelength of 345nm. The recorded decrease in absorbance is fitted using linear regression.">

<a class="fb" rel="rct" href="http://openwetware.org/images/9/9a/Nm_figure3.png" title="Reaction assay with conjugated enzyme. Continuous spectrophotometric measurement at a wavelength of 345nm. The recorded decrease in absorbance is fitted using linear regression.">

<a class="fb" rel="rct" href="http://openwetware.org/images/0/0b/UPLC_enzyme.PNG" title="UPLC measurements. The substrate and the products can be distinguished by their different retention times. A. Chromatogram of the UPLC measurement of the substrate. B. Chromatogram of the products obtained with the conjugated enzyme reaction. C. Chromatogram of the products obtained with the unmodified enzyme reaction. It can be observed that in the case of the conjugated enzyme both peaks are present, which is due to the decrease in enzymatic activity after conjugation to the fluorescent dye.">

<p>The DNA origami channel was incorporated during the polymersome preparation and was characterized using TEM and Cryo-TEM.</p>

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<p>For the tri-block copolymer, the polymersome formation was not affected by the addition of the DNA origami channel, and the DLS analysis confirmed that the polymersome size distribution was the same. In some of the polymersomes, particles across the membrane were observed. This indicates that the origami channels were successfully incorporated.</p>

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<p>To further confirm the incorporation of the channel in the wall, gold nanoparticles can be attached to the DNA channel. This will make them more distinguishable in the TEM and cryo-TEM analysis. This is planned as a follow-up experiment.</p>

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<p>The functionality of the channel was tested through a dye release assay. As a further confirmation of the incorporation of the channel in the membrane, and its functionality, we performed a dye release assay.</p>

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<p>For the di-block copolymer, the polymersome formation seemed to be affected by the presence of the DNA origami channel. The polymersome membrane surface became rough and also its stability decreased.The polymersomes had a smaller diameter than previously observed and were present in a smaller amount. This difficulted the observation of the DNA channel incorporation.</p>

<a class="fb" rel="channel" href="http://openwetware.org/images/3/3d/Nm_collage_3.png" title="Cryo-TEM images of Di-block polymersomes in presence of DNA channel with cholesterol modification (scale bar = 200nm). The number of polymersomes were smaller than previously observed and also present in a lower amount.">

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<img src="http://openwetware.org/images/b/b9/Dbdnachol.png" alt="" />

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</a>

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<figcaption>Di-block DNA-chol </figcaption>

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</figure>

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<figure class="box " >

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<a class="fb" rel="channel" href="http://openwetware.org/images/a/a9/Nm_collage_4.png" title="Cryo-TEM images of di-block polymersomes in presence of DNA channel with palmitate and archidic modification (scale bar = 200nm). The number of polymersomes were smaller than previously observed and also distorted by morphology.">

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<img src="http://openwetware.org/images/d/db/Dbdnapa.png" alt="" />

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</a>

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<figcaption>Di-block DNA-chol </figcaption>

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</figure>

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</div>

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<div class="clear"></div>

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<h2>Dye release assay</h2>

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<div class="project_box">

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<p>In order to confirm the functionality of the origami channels and their successful integration into the polymersome walls, a dye release experiment was performed. The fluorescein dye was encapsulated into the polymersomes in presence and absence of the channels. Fluorescein molecular weight is 332.29 Da and it can pass through the polymer walls, although with some difficulties. Thus, the incorporation of the channel should facilitate the exit of the dye. All the samples were placed inside dialysis bags and the fluorescein concentration outside the polymersomes was measured by UV/vis spectroscopy.</p>

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<p>As a positive control, the release of fluorescein from the dialysis bag without polymersomes was also performed.</p>

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<p> Tri-block polymersomes show around 80% of release within 24h whereas tri-block polymersomes with DNA channels exhibit complete release of dye. This is a strong hint of the successful incorporation of DNA channel within the tri-block polymersome walls, and that those are functional and allow the fluorescein to go through.</p>

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<p> Di-block polymersomes with and without origami channels have similar release profile. The release of dye from the di-block polymersome was less than 50% which proves association of dye with polymer. It is in agreement with confocal LSM images presenting strong adhesion of the dye molecules with di-block polymer which may inhibit polymersome formation. In this case, thus, the dye-release assay cannot be used to prove the incorporation of the channels due to the interaction of the dye with the polymersome walls.</p>

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<p>After 24h, the samples were also imaged by C-LSM (Confocal Laser Scanning Microscopy).</p>

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<p> Tri-block Polymersomes without channels clearly trapped the dye molecules which could be observed in C-LSM images, whereas the polymersome with DNA channels released most of the dye and residual polymers containing dye could only be imaged with 10x higher laser power and increased gain. This shows that in the latter case, almost all the dye could go outside of the polymersomes.</p>

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<p> Di-block polymersomes were highly destabilized in presence of dye. Only a few polymersomes encapsulated dye molecules similar to tri-block polymersome. Most polymersome were in disrupted state and strongly adhering fluorescence dye molecules. This result is in agreement with cryo-TEM images were we observed the similar structure of polymersome. </p>

<a class="fb" rel="Dye assay" href="http://openwetware.org/images/2/2b/Confocal_dye_triblock.png" title="Confocal-LSM image for Tri-block Polymersome after 24hr Dialysis. On the left, the control of the polymersomes without channel shows dye still inside the polymersomes, whereas in the presence of both cholesterol-modified or palmitate-arachidic-acid- modified channels 10 times more laser power was needed to see at least some remainigs of the dye inside. ">

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<img src="http://openwetware.org/images/a/a1/Pol1_clsm.png" alt="" />

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</a>

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<figcaption>C-LSM of Tri-block polymersome</figcaption>

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</figure>

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<figure class="box " >

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<a class="fb" rel="Dye assay" href="http://openwetware.org/images/f/fa/Confocal_dye_diblock.png" title="Confocal-LSM image for Di-block Polymersome after 24hr Dialysis. On the left, the control of the dye encapsulated inside the polymersoes without DNA channels, which presents less incorporation than with the Tri-block polymersomes. With DNA channel modified with cholesterol and palmitate-archidic similar images were obtained.">

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<img src="http://openwetware.org/images/2/2c/Pol2_clsm.png" alt="" />

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</a>

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<figcaption>C-LSM of Di-block polymersome</figcaption>

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</figure>

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<div class="clear"></div>

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<h2>Incorporation of the enzyme</h2>

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<div class="project_box" >

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<p>The collagenase conjugated with the TAMRA fluorescent dye was encapsulated in both polymersomes. Dialysis was performed to eliminate the non-encapsulated enzyme. The encapsulation efficiency was calculated as the concentration after dialysis divided by the concentration before dialysis. For the Tri-block polymersomes, it was of around 70%, whereas for Di-block polymersomes, it resulted to be of around 30%.</p>

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<p>The samples were then imaged by C-LSM. Particles that correspond to the polymersomes with the encapsulated enzyme could be observed in both Tri-block and Di-block polymers.</p>

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<p>In order to prove that after dialysis all the free enzyme is removed, the polymerosmes were incubated with substrate solution and UPLC measurements were taken at different intervals of time. Even after 16 h of incubation, no products were detected, meaning that no free enzyme is present in the solution and that the enzyme is successfully encapsulated inside the polymersomes.</p>

<a class="fb" rel="Enzyme" href="http://openwetware.org/images/e/e4/Nm_uplc1.png" title="UPLC measurement to check for the presence of free enzyme after dialysis. A. Chromatogram of the products of the enzyme reaction performed with unmodified enzymes. B. Chromatogram of the substrate. C. Chromatogram of the reaction assay performed with di-block polymersomes, incubated for 16h. D. Chromatogram of the reaction assay performed with tri-block polymersomes, incubated for 16h">

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<h2>Incorporation of enzyme and channel</h2>

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<p>As we demonstrated that both DNA channel incorporation and enzyme encapsulation was successful. In focus to our Smart Nanoreactor with these dual functions, the assembly of DNA channels and encapsulation of active enzyme at the same time is to be demonstrated. We choose enzymatic analysis to prove chemical transformation by FALGPA substrates which enter the polymersome through integrated DNA channel and get converted into products which can be detected with high sensitivity using UPLC (Ultra Performance Liquid Chromatography).</p>

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<h3>Tri-block polymer</h3>

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<p>In presence of enzyme, the DNA channel incorporation into membrane of polymersome was found to be successful shown using cryo-TEM image. We hypotheses the integration of DNA channels into the polymersome membrane from image contrast modulations around the polymersome membrane which are definitely not present in absence of DNA channel or Enzyme. In addition, the channels were also found to be perpendicular attached to the polymersome surface which may or may not be associated with polymersomes. Further, confirmatory results can be obtained by three dimensional cryo-TEM image collection and reconstruction.</p>

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<p>In order to obtain further evidence for DNA channel incorporation into polymersomes, negative stained sample were inspected under TEM. Although one should consider that the polymersomes get at least partly collapsed during this preparation the staining of DNA channels with and without tri-block polymer shows significant morphology differences. In absence of polymer, the DNA channels are clearly visible as cylindrical motif with sharp defined contours. While in presence of polymer, the DNA channels are embedded in a diffuse matrix addressed as polymer shell which is attached to the channel.</p>

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<p>In order to confirm our hypothesis, enzymatic analysis was performed and analyzed using UPLC.</p>

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<p>As we observed in DNA channel incorporation, the di-block polymersome represented the similar morphology with highly distorted membrane almost present in fragments rather intact polymersomes.</p>

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<a class="fb" rel="enzyme_channel" href="http://openwetware.org/images/f/f6/DNAcholp1.png" title="Cryo-TEM images of incorporated DNA channel with cholesterol modification in Tri-block polymersomes in presence of enzymes. The green arrows indicates the dark pattern around polymersome membrane which corresponds to incorporation of the cholesterol-modified DNA channels.">

<a class="fb" rel="enzyme_channel" href="http://openwetware.org/images/6/6f/DNAcholp2.png" title="Cryo-TEM images of Di-block polymersomes in presence of DNA channel with cholesterol modification and enzyme. The polymersome formation is highly inhibited which can be seen due to aggregation and only few stable polymersome can be seen.">

<a class="fb" rel="enzyme_channel" href="http://openwetware.org/images/7/7f/DNApap2.png" title="Cryo-TEM images of Di-block polymersomes in presence of DNA channel with palmitate and archidic modification and enzyme. Morphology of polymersome is highly distorted due to presence of DNA channel and enzyme which can be observed.">

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<p>The enzymatic assay was performed to prove that our system works: the channel allows the transport of the substrate in and out of the polymersomes, the encapsulated enzyme is still active and the reaction takes place.</p>

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<p>The reaction assay was performed after dialysis of the samples, so that no free enzyme is present in the solution. For the samples with DNA channel, two types of reaction were set, one with higher concentration and another with lower concentration of the polymersomes, to distinguish which one is the best to observe the reaction. The reaction was performed and samples were taken at 3h, 8h30min and 48h. Those were frozen and the reaction products were analyzed by UPLC.</p>

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<p> For the tri-block polymersomes, conversion of substrate into product could already be observed after 3 h incubation, even in the samples with lower concentration. However, the reaction rate in these samples is slower because even after 48 h the majority of the substrate remained unconverted. Nevertheless, the reaction for the tri-block polymersomes could also be observed in the sample without channels. For this reason, this assay should be repeated with an optimization of the concentration of the enzyme and the dialysis time.</p>

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<p>Previous reactions with the polymersomes without channels and the enzyme encapsulated showed that there was no conversion of the substrate, so in this case the apparition of the product could be due to a too short dyalisis time.As a follow-up experiment, proteases will be added outside the polymersomes before the reaction assay in order to make sure that no free enzyme is present in the solution, thus proving that the enzyme inside the nanoreactors is protected and still active.</p>

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<p>In the case of di-block polymersomes, the samples showed no reaction, even with higher concentration of polymersomes. This correlates with the previous results that showed that the formation of these polymersomes was affected by the incorporation of the channel, and so no enzyme could be incorporated.</p>

<a class="fb" rel="int_rxn" href="http://openwetware.org/images/8/8c/Nanoreactors_diblock.PNG" title="Enzyme reaction assay with di-block polymersomes. A. Chromatogram of the substrate. B. Chromatogram of the di-block polymersomes without channel, incubated for 48h with the substrate solution. C. Chromatogram of the di-block polymersomes with channel, having 5’ palmitate and arachidic acid modifications, incubated for 48h with the substrate solution. D. Chromatogram of the di-block polymersomes with channel, having 3’ cholesterol modifications, incubated for 48h with the substrate solution..">

<p>Our project does not finish here. There is a set of planned experiments that due to the time constraint were impossible to perform. The ones that catch our interest are the following.</p>

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<h3>Quantifying the incorporation of the channels</h3>

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<p> We will attach gold nanoparticles of around 2-5 nm in size that have attached to their surface a ssDNA sequence that can hybridize with the scaffold of the DNA origami channel.</p>

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<p> This will help distinguish the DNA origami channel in the TEM and cryo-TEM imaging and so a quantification of the channels that are present in each polymersome will be made.</p>

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<h3>Structural studies of the origami channel</h3>

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<p> In collaboration with Prof. Dr. Rasmus Schroeder, in Heidelberg University, we will do structural studies of the DNA origami 3D structure in the nanometer scale.</p>

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<h3>Optimization of the Smart Nanoreactor</h3>

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<p>The formation of the Smart Nanoreactor will be optimized with regard to the concentration of enzyme, DNA channel and coblock polymers.</p>

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<p>Furthermore, reaction could be observed in the negative control. Previously to the reaction assay, proteases will be introduced outside the polymersomes that can degrade the enzymes that are not incorporated. Thus, we will prove that the observed products are due only to the encapsulated enzyme.</p>

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<p>In the case of the di-block polymersomes, their formation with the presence of the origami channel was affected. Thus, we will further investigate their formation with lower concentrations of the origami channel.</p>

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<h3>Antibody immobilization</h3>

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<p>For targeted delivery in the in-vivo studies, the coblock polymers will be functionalized with a suitable linker for the antibody attachment. Thus, we will be able to specifically target cells with our Smart Nanoreactor.</p>

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<h3>In-vitro and in-vivo studies</h3>

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<p>In-vitro studies with cell cultures will be performed to test the delivery of the antibody-modified nanoreactors to the desired cells.</p>

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<p>Furthermore, in collaboration with Prof. Dr. Michael Brand in CRTD, we will perform in-vivo studies using Zebrafish, because due to their transparent body, we will be able to easily trace the Smart Nanocontainer and analyze its efficiency in targeting.</p>

<p>In recent years DNA origami reached incredible popularity in nanotechnology due to its vast design possibilities and its broad potential applications. While having numerous advantages it is still exceptionally expensive to produce in high amounts for mass applications, whereas inorganic polymers, another range of materials available for biomolecular design can compensate for this disadvantage. Nowadays they can be found in almost every aspect of our lives, in medicine and technology through their wide range of different functionalities and their moderate price. In our project we aim to combine those two artificial materials into one device: functional and responsive polymers as carrier material with highly flexible designable DNA origami for more complex access points to gain the best of both of them.</p>

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<p>Our main application presented here in our video we describe using our Smart Nanoreactor as a new form of drug delivery system, which takes great advantages of the polymersome's role as carrier for multiple different other functional components. In chemotherapy a big range of treatment methods use drugs which are damaging both cancerous as well as healthy tissue, putting the patient in a high stress during the treatment. But with out Smart Nanoreactor we offer a solution for this problem. As a first step the Nanoreactor is injected into the patient and through antibodies immobilized on the outside of the sphere, the device is targeting specific pathogenic tissue like cancer and is localized only in this area. In the second step an inactive form of a pro-drug gets systematically introduced into the patient, so it is localized in healthy as well as pathogenic tissue. Now the pro-drug can enter the Nanoreactor through the origami channels and the encapsulated enzyme can convert the inactive pro-drug to its active state in which it is damaging both healthy and pathogenic tissue. After exiting the Nanoreactor again the active drug can interact with its surroundings, due to the location of the Nanoreactor the closest cells are the intended cancerous tissue. After the targeted cells are treated the immobilized antibodies loose their binding partner, the reactor dis-attaches and is filtered out of the body through the kidneys.</p>

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<a class="fb" rel="enzyme_channel" href="http://openwetware.org/images/f/fb/DNApap1.png" title="Cryo-TEM images of incorporated DNA channel with palmitate and archidic modification in Tri-block polymersomes in presence of enzymes. The green arrows indicates the dark pattern around polymersome membrane which corresponds to incorporation of the palmitate and arachidic acid-modified DNA channels.">

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<h2>pH-responsive Nanocontainer</h2>

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<p>Another powerful feature of our device is the use of a pH-responsive polymer, which enables us to add even more functionality to our Nanoreactor. The described Di-block copolymer has tight and stable walls in basic pH but gets leaky in acidic milieu. We could use this to create another route of transport for molecules that are too big to fit through the origami channel. In high pH the container circles through its environment and takes up small molecules from the outside, an enclosed enzyme crosslinks them together until they are too big to exit again through the channel. After the container reaches surroundings with lower pH, its walls become more porous and allow the exit of its cargo through pores in the polymersome. A great advantage is the reversibility of this process, so when the vesicle is empty, it can be brought back to basic pH, the walls are sealed tight again and the reactor can take up more substrate through the DNA channel.</p>

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<p>This feature could not only open a variety of applications in medicine where our device can react to physiological changes in pH but also in technology and environmental engineering for example. Synthesizing processes can be automated and our Nanoreactor can respond to different stages of a pH changing reaction or it could be used to clean freshwater supplies via independent sampling of contaminated wells.</p>

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<h2>Smart Nanofactory</h2>

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<p>The possibilities of our device for technological applications do not end here but observing nature and discovering the formation of polymersomes inside bigger polymersomes during Cryo-TEM analysis another great range of technical applications came to our minds. If we extend this process to creating complete Smart Nanoreactors encapsulated in other Nanoreactors we can create whole Nanofactories in the future, similar to the different lipid vesicle compartments in living cells like vacuoles.</p>

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<p>Combining different reactors with various different DNA origami channels, polymersomes and enzymes we are able to design a system, which can independently catalyze complex chains of reactions. Immobilized targeting proteins on the outside of polymersomes and their sizes act for sorting of the reactors, while the different channels determine the order of entry that the substrates have to use to complete the full chain of a reaction. Besides easy separation of product molecules form enzymes, our device has the advantage of formation of big structures by different enzymes in a complex order, one can even create repeats and loops by incorporating a range or Nanoreactors within each other, all of it independently.</p>

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<p>This was just a small trip through the wide spectrum of applications for our Smart Nanoreactor, but there is far more possible in the future. And all of this just with a plastic ball.</p>

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<a class="fb" rel="enzyme_channel" href="http://openwetware.org/images/a/ac/Nanofactory_1.png" title="Combining different reactors with various different DNA origami channels, polymersomes and enzymes we are able to design a system, which can independently catalyze complex chains of reactions.">

Revision as of 14:20, 26 October 2013

How to cure cancer with a plastic ball?

Check our video!

Welcome

we, the Dresden Nanormous, use polymers to produce hollow nanoreactors or polymersomes, whose walls contain channels for controlled transport. These channels are built by the DNA-origami technique that way we produce a highly flexible system. By encapsulation of an enyzme and the addition of antibodies we create a smart nanoreactor which could be used to fight cancer!

Polymersome

DNA origami channel

Internal reaction

The picture shows a SEM image of our first polymersomes! The average diameter is about 200nm.